Photochemistry and Photobiology, zyxwvuts 1998, 67(6): 676-682 zyxwvu Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dar k-G rown Leaves Philippe Eullaffroy', Radovan Popovic' and Fabrice Franck** 'Department of Chemistry, University of Quebec in Montreal, Montreal, Quebec, Canada and 2Laboratory of Photobiology, Department of Plant Biology, University of Liege, LiegeEart-Tilman, Belgium Received 16 December 1997; accepted 10 March 1998 ABSTRACT zyxwvutsrq The fluorescence yield of chlorophyll(ide) (Chlride])ex- cited by weak modulated light was recorded at room temperature during a zyxwvuts 2 h period after a short actinic light pulse that transformed all photoactive protochlo- rophyllide in dark-grown barley leaves. A typical pattern of fluorescence yield variations was found whatever the age of the leaf but with age-dependent changes in rates. Its successive phases were related to the Chl(ide) spectral shifts observed in low-temperature emission spectra. The fluorescence yield started at a high level and strongly de- clined during the formation of Chlide,,, from Chlide6ss within a few seconds. It increased to a transient maxi- mum during the Shibata shift (15-25 min) that resulted in Chl(ide),,,. zyxwvutsrq A final, slow decrease to a steady state oc- curred during the final red shift to ChI6,,. Pretreatments with 6-aminolevulinic acid, chloramphenicol or 1,lO- phenanthroline resulted in correlated modifications of Chl(ide) fluorescence yield transients and shifts of the low-temperature Chl(ide) emission band. The complex response of the final decrease phase of the fluorescence yield to these compounds suggests that it results both from the assembly of photosynthetic Chl proteins and from the reorganization of the etioplast membrane sys- tem. From these results it is concluded that continuous recordings of Chl(ide) fluorescence yield after a short light pulse represent a useful tool to monitor the kinetics of pigment-protein organization and primary thylakoid assembly triggered by Pchlide photoreduction. INTRODUCTION Protochlorophyllide (Pchlide) photoreduction is the photoen- zymatic step that makes chlorophyll (Chl) biosynthesis a *To whom correspondence should be addressed at: Laboratory of Photobiology, Department of Botany B22, University of Litge, B-4000 Libge/Sart-Tilman, Belgium. Fax: 3243662926; e-mail: F.Franck@ulg.ac.be zyxwvutsrqpon tAbbreviutions; Chl(ide),, chlorophyll or chlorophyllide zyxwvutsr u with a low-temperature emission maximum at X nm; &-ALA, &amino- levulinic acid; Pchlfide),, protochlorophyll or protochlorophyllide zyxwvut a with a low temperature emission maximum at X nm; POR, light-dependent NADPH : protochlorophyllide oxidoreductase; PSI, PSII, photosystem I or 11. 0 1998 American Society for Photobiology 0031-8655/98 $5.00+0.00 light-dependent process in higher plants. This reaction is cat- alyzed by the light-dependent NADPH : protochlorophyllide oxidoreductase (POR; EC 1.3.1.33) (1,2). In etioplasts of dark-grown leaves, POR-substrates complexes accumulate in prolamellar bodies and prothylakoids. A short light pulse triggers the phototransformation of these complexes in vivo, which in turn initiates a complex series of light-independent molecular and structural events in the etioplast inner mem- brane system. The rapid release of Chlide and its esterifi- cation to Chl a, mediated by Chl-synthase, triggers the ac- cumulation of plastid proteins and their assembly into func- tional photosynthetic core complexes (3). These processes initiate light-dependent chloroplast differentiation in angio- sperms. They are closely associated with a reorganization of the membrane system by the dispersal of prolamellar bodies and formation of primary photosynthetic membranes (4,s). Because Pchlide is the main red-light-absorbing pigment in dark-grown leaves, spectroscopy has been extensively used to monitor the above processes in vivo (2,3,6). Low- temperature fluorescence spectroscopy has been a sensitive method to detect different Pchl(ide) and Chl(ide) forms as- sociated with the different steps of etioplast phototransfor- mation by a short light pulse (6). Two Pchl(ide) emission bands at 633 and 655 nm are always detected in dark-grown leaves. The 655 nm band arises from POR-substrates com- plexes, identified in vivo as photoactive PChhde6,,. The 633 nm band is emitted by photoinactive pigments most probably unbound to POR (Pchl[ide],,,). The first Chlide products of Pchlide6,, phototransformation are POR : Ch1ide:NADP' complexes corresponding to the Chlide6,, spectral form. Through substitution of NADP+ by NADPH Chlide,,, is formed within some seconds (a minor fraction shifts to short- wavelength Chlide675, presumably a free pigment form). Fur- ther incubation in darkness results within minutes in the Shi- bata shift (7) with Chlide,,, as end product. Esterification of Chlide and prolamellar body dispersal closely overlap this shift. The final dark spectral step after one flash in vivo is a slow red shift of only 2-3 nm, during which Ch16,, of pho- tosystem I1 (PSII) develops within about 2 h in correlation with potential photosynthetic activity (8). The time-course of photoactive Pchlide regeneration overlaps the Shibata shift and the final red shift (9). The above spectral shifts were deciphered by comparing the emission spectra of samples that were frozen to liquid 676